A recombinase polymerase amplification-based lateral flow strip assay for rapid detection of genogroup II noroviruses in the field.

Human norovirus is the leading cause of viral gastroenteritis worldwide. Rapid detection facilitates management of disease outbreaks, but field diagnosis is difficult to achieve due to the lack of reliable and portable methods. Recombinase polymerase amplification (RPA) is a robust isothermal amplification method that is capable of rapidly amplifying and detecting nucleic acids using simple equipment. In this study, RPA combined with lateral flow (LF) strips specific for human genogroup II (GII) noroviruses was established and evaluated. The assay specifically detects purified GII noroviruses as well as RNA in boiled human stool samples, with a sensitivity of 50 norovirus genome copies per reaction. The whole detection procedure of the one-step RT-RPA-LF is completed within 20 min, which is eight times faster than that of the standard real-time RT-PCR. The RT-RPA-LF method described here is suitable for rapid field diagnosis of all GII noroviruses in human stool samples.

Structural dynamics of the CROPs domain control stability and toxicity of Paeniclostridium sordellii lethal toxin.

Paeniclostridium sordellii lethal toxin (TcsL) is a potent exotoxin that causes lethal toxic shock syndrome associated with fulminant bacterial infections. TcsL belongs to the large clostridial toxin (LCT) family. Here, we report that TcsL with varied lengths of combined repetitive oligopeptides (CROPs) deleted show increased autoproteolysis as well as higher cytotoxicity. We next present cryo-EM structures of full-length TcsL, at neutral (pH 7.4) and acidic (pH 5.0) conditions. The TcsL at neutral pH exhibits in the open conformation, which resembles reported TcdB structures. Low pH induces the conformational change of partial TcsL to the closed form. Two intracellular interfaces are observed in the closed conformation, which possibly locks the cysteine protease domain and hinders the binding of the host receptor. Our findings provide insights into the structure and function of TcsL and reveal mechanisms for CROPs-mediated modulation of autoproteolysis and cytotoxicity, which could be common across the LCT family.

Updating a New Semi-nested PCR Primer Pair for the Specific Detection of GII Norovirus in Oysters.

Oysters are major transmission vectors of noroviruses (NoVs) in the environment. Outbreaks of NoVs are often associated with the consumption of NoV-contaminated oysters. Laboratory confirmation of suspected oyster samples is a critical step in the surveillance and control of NoVs. Because of non-specific amplification, false-positive results are frequently obtained by semi-nested RT-PCR with the presently widely used primer set (G2SKF/G2SKR). Here, a novel universal PCR primer set N (NG2OF/NG2OR) specific for genogroup II (GII) NoVs was designed based on all GII NoV sequences available in public databases. Specific products were obtained with the primer set N when the NoV-positive oysters, spiked with each of five representative genotypes of GII NoVs (GII.17, GII.13, GII.4, GII.3, and GII.12), were subjected to analyzing. No products were detected with the primer set N for the NoV-negative oysters, while the primer set C gave various non-specific bands. Twenty-three out of 156 fresh oyster samples were NoV-positive with both the primer set N and the classic primer set, while eight were NoV-positive solely with the primer set N. Compared with the classic primer set, the newly designed primer set N had a higher detection rate and improved specificity for GII NoVs in oyster samples. These results show that the novel PCR primer pair is specific and applicable for the detection of GII NoVs in oysters.